HBV vectors and cells for producing the same

ABSTRACT

This invention relates to an HBV vector in which functional genes of HBV are at least partially deleted. In addition, this invention concerns a process for producing such an HBV vector as well as cells usable for this purpose.

HBV Vectors and Cells for Producing the Same This invention relates to HBV vectors, processes for the provision thereof and cells usable for this purpose as well as the use of the HBV vectors.

Efficient methods are required for a gene therapy to transfer a “therapeutic” DNA into select target organs. Up to the present, retroviral vectors have been used above all for this purpose. However, they have the drawback that the cells to be treated first have to be propagated in vitro, infected and then be transferred into the patient again. However, it should be the objective of a gene therapy to treat cells in situ.

For an in vivo gene therapy the strict organ specificity of the employed vector system is an absolute precondition. The hepatocytes of the liver are of special interest for this purpose. The liver is the source of most of the serum proteins and plays a central part for the regulation of the metabolism in the peripheral organs. Therefore, a plurality of different, inherited metabolic defects manifest themselves in the liver. In addition, the liver is also affected in the case of some viral infections which can be treated only very poorly. Furthermore, the liver could be used as a bioreactor for the secretion of diverse proteins, provided that a suitable gene transfer system existed. Thus, new paths could be trodden also for the treatment of diseases which do not manifest themselves in the liver.

However, a liver cell-specific gene transfer system for which an in vivo application could be in consideration does not yet exist.

Therefore, it is the object of the present invention to provide a gene transfer system which is liver cell-specific and suitable for an in vivo gene therapy.

According to the invention this is achieved by the subject matters in the claims.

Therefore, the subject matter of the present invention relates to a HBV vector in which functional genes of HBV are at least partially deleted.

The expression “HBV” refers to hepatitis B virus. This is a DNA virus having a genome length of 3.2 kb. The genome of hepatitis B virus contains four partially overlapping open reading frames (ORF): the pol-ORF (HBV polymerase), the S-ORFs (surface proteins), the C-ORFs (capsid proteins) and the X-ORF (viral transactivator) Hepatitis B virus is liver cell-specific.

The expression “HBV vector” comprises any HBV vector which is suitable for a gene transfer, especially in a gene therapy, most especially in an in vivo gene therapy. In this connection, the expression “vector” relates to a DNA molecule as well as a virus particle.

The expression “in which functional genes of HBV are at least partially deleted” refers to the fact that in an HBV vector according to the invention one to all genes necessary for the replication of HBV are partially or fully deleted. Such genes are especially those which code for polymerase, the surface proteins and the capsid proteins of HBV. Because of the above deletion, an HBV vector according to the invention can no longer replicate independently in a eukaryotic cell.

It is advantageous when in an HBV vector according to the invention the genes for the polymerase, the surface proteins and the capsid proteins of HBV are at least partially deleted. It is especially advantageous when these genes are fully deleted.

Furthermore, it is of advantage when in an HBV vector according to the invention the gene of the transactivator of HBV is also mutated or partially deleted and fully deleted, respectively.

A preferred HBV vector of the present invention is pHBV/V1. Its DNA sequence is indicated in FIG. 1. In pHBV/V1, the genes for the polymerase, the surface proteins and the capsid proteins of HBV are fully deleted. Likewise, the gene for the transactivator of HBV has an ochre mutation. pHBV/V1 was deposited with the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [German-type collection of micro-organisms and cell cultures]Mascheroder Weg 1b, D-38124 Braunschweig, Germany) under DSM 9947 on May 3, 1995.

Because of its deletion, an exogeneous or foreign or heterologous DNA can be inserted in an HBV vector according to the invention, and it can then be expressed in the cells accommodating the HBV vector. A foreign DNA may be any DNA, particularly a diagnostically and/or therapeutically effective gene. The length of the foreign DNA may vary, it being advantageous when it does not exceed about 3 kb. On a protein basis, this length corresponds to a molecular weight of over 100,000, which is quite sufficient for gene-therapeutic applications.

A foreign DNA is inserted in an HBV vector according to the invention via the “multiple cloning site” of the latter. Hence it is also possible to insert the foreign DNA between two inverse terminal repetitions of adeno-associated viruses. This would increase the integration frequency as well as the integration specificity of the foreign DNA in a special chromosome.

Another subject matter of the present invention relates to a process for producing the above HBV vectors. In such a process, the defect in the independent replication of an HBV vector according to the invention is overcome by transfecting it into cells which express functional HBV proteins. The expression of the HBV proteins can be transient and/or stable, a stable expression being preferred. HBV vectors are provided as DNA molecules as well as virus particles by the process according to the invention.

Common methods can be used for producing the above cells. It is favorable to transfect hepatoma cells, e.g. Hep G2 cells (cf. Knowles, B. B. et al., Science 209 (1980), 497-499) with expression plasmids coding for functional HBV proteins. It is especially favorable when the genes for the individual functional HBV proteins are present on differing expression plasmids.

For the preparation of the above expression plasmids, it proves to be favorable to use common HBV vectors having selection markers and delete therein the epsilon region necessary for packaging as well as differing functional HBV genes. The DNA sequence of HBV, including the epsilon region, is known (cf. e.g. Fujiyama, A. et al., Nucl. Acids Res. 13, (1983), 4601-4610; Polack, J. R. and Ganem, D., J. Virol. 67, (1993), 3254-3263).

Common methods can be used for the transfection of hepatoma cells, e.g. Hep G2 cells, having the above expression plasmids. For example, a DEAE-dextran process (cf. McCutchan, J. H. and Pagano, J. S., J. Natl. Cancer Inst. 41, (1968), 351-357) is suitable for a transient expression of the functional HBV proteins, whereas e.g. a calcium phosphate precipitation process (cf. Graham, F. L. and van der Eb, A. J., Virology, 52 (1973), 456-467) has to be mentioned for a stable expression. Cells are obtained which express functional HBV proteins. Such cells also represent a subject matter of the present invention. Of these those are preferred which express the polymerase, the surface antigens and the capsid proteins of HBV, particularly express them in stable fashion.

The present invention serves for providing a gene transfer system which is liver cell-specific and suitable for a gene therapy, particularly an in vivo gene therapy. The gene transfer system comprises HBV vectors and cells in which these vectors can be provided.

The present invention enables to transfer foreign DNA into liver cells where it is expressed. Thus, it does not only open up possibilities of treating monogenic metabolic defects, e.g. familial hypercholesterolemia, hyper-ammonemia, hyperbilirubinemia, phenylketonuria, α₁-antitrypsin deficiency, hemophilia, etc., but also of treating multifactorial diseases such as the virus hepatitises, e.g. HBV, HCV, HDV, and last but not least the primary liver cell carcinoma.

In addition, the present invention provides the possibility of using the liver as bioreactor for the secretion of any therapeutic proteins into the blood. This results in new aspects of gene therapy, which exceed by far the original target organ, such as the treatment of malignant diseases, of viral infections or generally of diseases which do not manifest themselves in the liver.

Moreover, by means of the present invention it ispossible to monitor the most differing processes for killing viruses in body fluids withdrawn, e.g. blood. For this purpose, an HBV vector according to the invention, which is provided with a minotor gene, can be added to the body fluid before the process starts and then be determined at certain intervals.

Thus, the present invention is perfectly suited as a reagent for diagnosis and/or treatment.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows the DNA sequence of an HBV vector according to the invention, pHBV/V1. This DNA sequence comprises the following:

Nucleotide No. Elements 3262-3272 “direct repeat II” of HBV 3496-3504 “direct repeat I” of HBV 3519-3579 epsilon region of HBV 3694-3748 “multiple cloning site” 3962-3970 “direct repeat II” of HBV 4196-4204 “direct repeat I” of HBV 4288-4293 poly-A region

The other nucleotides comprise those of the cloning vector pSPT 19 (cf. Example 1).

The following examples explain the invention.

EXAMPLE 1 Construction of an HBV Vector According to the Invention, pHBV/V1

A 621 bp long BamHI/Stu I fragment of adr 4 was cut out of the plasmid pBRHBadr4 (cf. Fujiyama, A. et al., above) which contains an HBV subtype, adr 4, and inserted in the cloning vector PSPT 19 opened by BamHI/Sma I (cf. catalog of Boehringer Mannheim, order No. 909815). The plasmid PSPT 0.2×HBV was obtained. This plasmid contains all regulatory elements of the E₁₁/C_(p) region, which are necessary for the HBV replication. In pSPT 0.2×HBV, a complete 3215 bp long HBV genome (BamHI/BamHI fragment) of pBRHBadr4 (see above) was inserted at the BamHI restriction site. The plasmid pSPT 1.2×HBV was obtained. The above, regulatory elements of the E₁₁/C_(p) region are present in this plasmid at the 5′ end and also at the 3′ end of the HBV portion. In addition, an ochre mutation in codon 8 was inserted in the X-ORF (see above) of pSPT 1.2×HBV. The plasmid PSPT 1.2×HBV Mx was obtained. This plasmid was cleaved with StuI and NcoI (restriction sites in the HBV genome) and the functional genes of HBV were removed. A “multiple cloning site” was inserted instead. An HBV vector according to the invention, pHBV/V1, was obtained.

EXAMPLE 2 Expression of a Foreign DNA in an HBV Vector According to the Invention, pHBV/V1

A known foreign DNA was inserted in the “multiple cloning site” of pHBV/V1 of Example 1. This was the luciferase gene and the LacZ gene fragment, respectively. In the former case, the plasmid pV1/HBV-Luc was obtained and, in the latter case, the plasmid pV1/HBV-LacZ was obtained. Both plasmids were used for a transient transfection of HepG2 cells (see above).

It showed that both foreign DNAs were expressed. This is also an evidence for the replication of pHBV/V1 in cells which produce HBV-WT particles.

1 1 4421 DNA Artificial Sequence Description of Artificial Sequence pHBV/V1 1 tatagtgtca cctaaatcgt atgtgtatga tacataaggt tatgtattaa ttgtagccgc 60 gttctaacga caatatctac aagcctaatt gtgtagcatc tggcttactg aagcagaccc 120 tatcatctct ctcgtaaact gccgtcagag tcggtttggt tggacgaacc ttctgagttt 180 ctggtaacgc cgtcccgcac ccggaaatgg tcagcgaacc aatcagcagg gtcatcgcta 240 gccagatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg 300 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca 360 tgagcgcttg tttcggcgtg ggtatggtgg caggcccgtg gccgggggac tgttgggcgc 420 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact 480 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgatatggtg cactctcagt 540 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac 600 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 660 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 720 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 780 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 840 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 900 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 960 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 1020 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 1080 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 1140 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 1200 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 1260 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 1320 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 1380 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 1440 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 1500 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 1560 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 1620 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 1680 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 1740 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 1800 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 1860 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 1920 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 1980 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 2040 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 2100 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 2160 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 2220 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 2280 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa 2340 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 2400 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 2460 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 2520 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 2580 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 2640 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 2700 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 2760 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatata 2820 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc 2880 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 2940 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcccag 3000 ctggcttatc gaaattaata cgactcacta tagggagacc caagcttgca tgcctgcagg 3060 tcgactctag aggatcctgc gcgggacgtc ctttgtctac gtcccgtcgg cgctgaatcc 3120 cgcggacgac ccgtctcggg gccgtttggg actctaccgt ccccttcttc atctgccgtt 3180 ccggccgacc acggggcgca cctctcttta cgcggtctcc ccgtctgtgc cttctcatct 3240 gccggtccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac cgtgaacgcc 3300 caccaggtct tgcccaaggt cttacataag aggactcttg gactctcagc gatgtcaacg 3360 accgaccttg aggcatactt caaagactgt ttgtttaagg actgggagga gttgggggag 3420 gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt ctgttcacca 3480 gcaccatgca actttttcac ctctgcctaa tcatctcatg ttcatgtcct actgttcaag 3540 cctccaagct gtgccttggg tggctttggg gcatggacat tgacccgtat aaagaatttg 3600 gagcttctgt ggagttactc tcttttttgc cttctgactt ctttccttct attcgagatc 3660 tcctcgacac cgcctcagct ctatatcggg aggcctgcgg ccgctcgagt taactagtcg 3720 cgatgcatcg atgatcaccc gggccatggc tgctagggtg tgctgccaac tggatcctgc 3780 gcgggacgtc ctttgtctac gtcccgtcgg cgctgaatcc cgcggacgac ccgtctcggg 3840 gccgtttggg actctaccgt ccccttcttc atctgccgtt ccggccgacc acggggcgca 3900 cctctcttta cgcggtctcc ccgtctgtgc cttctcatct gccggtccgt gtgcacttcg 3960 cttcacctct gcacgtcgca tggagaccac cgtgaacgcc caccaggtct tgcccaaggt 4020 cttacataag aggactcttg gactctcagc gatgtcaacg accgaccttg aggcatactt 4080 caaagactgt ttgtttaagg actgggagga gttgggggag gagattaggt taaaggtctt 4140 tgtactagga ggctgtaggc ataaattggt ctgttcacca gcaccatgca actttttcac 4200 ctctgcctaa tcatctcatg ttcatgtcct actgttcaag cctccaagct gtgccttggg 4260 tggctttggg gcatggacat tgacccgtat aaagaatttg gagcttctgt ggagttactc 4320 tcttttttgc cttctgactt ctttccttct attcgagatc tcctcgacac cgcctcagct 4380 ctatatcggg agggggtacc gagctcgaat tccgtgtatt c 4421 

What is claimed is:
 1. A hepatitis B virus (HBV) vector comprising a modified HBV genome characterized by a mutated HBV-X-ORF rendering the HBV-X-ORF non-functional by virtue of a mutation in codon 8 of the HBV-X-ORF; and at least one deletion of pol-ORF, S-ORF and C-ORF which renders the HBV genome replication-defective.
 2. A hepatitis B virus (HBV) vector comprising a modified HBV genome characterized by (i) an HBV-X-ORF including a modification rendering the HBV-X-ORF non-functional, wherein the modification is a partial deletion of the HBV-X-ORF; and (ii) a deletion of at least one of pol-ORF, S-ORF and C-ORF.
 3. The HBV vector deposited on May 3, 1995 with Deutsche Sammlung von Mikroorganismen und Zelikulturen under accession number DSM
 9947. 4. A hepatitis B virus (HBV) vector comprising a modified HBV genome characterized by (i) an HBV-X-ORF including a modification rendering the HBV-X-ORF non-functional, wherein the modification is a partial deletion of the HBV-X-ORF and (ii) a deleted pol-ORF, S-ORF and C-ORF open reading frames.
 5. A hepatitis B virus (HBV) vector comprising a modified HBV genome including the following modifications: (a) a modification which renders nonfunctional the HBV-X-ORF, consisting of a mutation in codon 8 of HBV-X-ORF; and (b) deletions of the pol-ORF, S-ORF and C-ORF open reading frames. 